Treatment of prostate cancer by inhibitors of ATP synthase

ABSTRACT

The present invention is directed to a method of treating or preventing prostate cancer in a subject in need of such a treatment by administering an inhibitor of ATP synthase activity in a pharmaceutically effective amount. The present invention also provides for a pharmaceutical composition comprising an inhibitor of ATP synthase.

[0001] This application claims the benefit of priority of the U.S.provisional application U.S. Ser. No. 60/329,178 filed Oct. 11, 2001 andthe U.S. provisional application U.S. Ser. No. 60/331,965, filed Nov.21, 2001, each of which is incorporated by reference in its entirety.

FIELD OF INVENTION

[0002] This invention concerns methods for treating or preventingprostate cancer by inhibitors of ATP synthase activity.

BACKGROUND OF THE INVENTION

[0003] Prostate cancer is the most common cancer in men with anestimated 244,000 cases in 1995 in the United States. It is the secondleading cause among men who die from neoplasia with an estimated 44,000deaths per year. Prompt detection and treatment is needed to limitmortality caused by prostate cancer. As described in W. J. Catalona,“Management of Cancer of the Prostate,” (New Engl. J. Med. 331(15):996-1004 (1994)), the management of prostate cancer can be achieved bywatchful waiting, curative treatment, and palliation.

[0004] A number of approaches have been developed to treat prostatecancer. Where prostate cancer is localized and the patient's lifeexpectancy is 10 years or more, radical prostatectomy offers the bestchance for eradication of the disease. Historically, the drawback ofthis procedure is that most cancers had spread beyond the bounds of theoperation by the time they were detected. After surgery, if there aredetectable serum prostate-specific antigen concentrations (PSA),persistent cancer is indicated. In many cases, prostate-specific antigenconcentrations can be reduced by radiation treatment. However, thisconcentration often increases again within two years.

[0005] Cytotoxic chemotherapy is largely ineffective in treatingprostate cancer. Its toxicity makes such therapy unsuitable for elderlypatients. In addition, prostate cancer is relatively resistant tocytotoxic agents.

[0006] In view of the deficiency of the existing treatment approaches,it is of great significance to pursue new methods of treatment thatparticularly target the prostate tumor cells, such as anti-prostatetumor agents. The present invention is intended to use inhibitors of ATPsynthase activity as such anti-tumor agents.

[0007] ATP synthase is the enzyme catalyzing the synthesis of ATP. It isseen as spherical projections from the inner membrane surface ofmitochondria, consisting of two subcomplexes: F₀ and F₁. F₁ consists offive different polypeptide chains with the stoichiometry α₃β₃γδε. The F₀subcomplex 11 different subunits and forms a hydrophobic unit that spansthe inner mitochondrial membrane. The F₀ complex has the ability totranslocate protons across the membrane from their high potential on theoutside, and when coupled to the F₁ subcomplex, ATP synthesis can beachieved. (Harold, F. M., The Vital Force: A Study of Bioenergetics (W.H. Freeman, 1986), pp. 238) (which is hereby incorporated by reference).

[0008] Apart from the traditional role in ATP production, recent studiessuggest that ATP synthase may play critical roles in tumor cellmetastases. Studies have shown that ATP synthase plays important rolesin angiogenesis (the generation of new blood vessels required by tumorto expand beyond a prevascular size). Angiostatin, a potent antagonistof angiogenesis, was found to bind ATP synthase on the surface of humanendothelial cells (Moser, T. L., et al., Proc. Natl. Acad. Sci. USA:Vol. 96, pp 2811-2816, 1999). These authors propose that all componentsof F₁ ATP synthase catalytic core are present on the endothelial cellsurface since the complex is able to synthesize ATP. In addition, thesurface-associated enzyme is active in ATP synthesis as shown bydual-label TLC and bioluminescence assays. ATP synthase activities ofthe enzyme are inhibited by angiostatin as well as by antibodiesdirected against the α- and β-subunits of ATP synthase in cell-based andbiochemical assays. These experimental results suggest that theinhibitors of ATP synthase activity, such as antibodies against ATPsynthase, function as antagonists of angiogenesis. (Moser, T. L., etal., Proc. Natl. Acad. Sci. USA: Vol. 98, pp 6656-6661, 2001). Inaddition, β-subunit of ATP synthase is also found to differentiallyexpress on the surface of some tumor cell lines. It is speculated thatATP synthase is an important ligand in the effector phase of a cytolyticpathway, and plays a role in lymphocyte-induced tumor destruction. (Das,B., et al., J. Exp. Med. Vol. 180, pp 273-281, 1994).

[0009] So far no investigation has been conducted to explore thepossible therapeutic application of inhibitors of ATP synthase inprostate cancer treatment. This invention is directed to methods fortreating and preventing prostate cancer by using the inhibitors of ATPsynthase.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to a method of treating orpreventing prostate cancer in a subject in need of such a treatment byadministering an inhibitor of ATP synthase in a pharmaceuticallyeffective amount. The present invention also provides for apharmaceutical composition comprising an inhibitor of ATP synthase,preferably, said inhibitor is an anti-ATP synthase antibody

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1. Comparison of lipid rafts prepared from normal prostatecells and three prostate carcinoma cell lines, DU 145, LNCaP, and PC-3.Equal amounts of protein from lipid raft preparations were separated bySDS-PAGE and then visualized by silver staining. Differentiallyexpressed proteins are marked.

[0012]FIG. 2. Comparison of lipid rafts prepared from LNCaP and normalprostate cells by 2-dimensional electrophoresis. Equal amounts ofprotein from lipid raft preparations were separated by 2-dimensionalelectrophoresis and then visualized by silver staining. Protein spotsthat are present in the LNCaP sample, but not the normal prostate sampleare denoted with arrows. Protein spots that have been identified bypeptide mass profiling are labeled with numbers (see Table 1).

[0013]FIG. 3. ATP synthase is present in lipid rafts preparations fromprostate cancer cell lines, but not from normal prostate cells. Equalamounts of lipid raft proteins prepared from normal prostate cells andthree prostate carcinoma cell lines (LNCaP, DU 145, and PC-3) wereseparated by SDS-PAGE and then electrotransferred onto a PVDF membrane.Western blotting was performed using an antibody specific for ATPsynthase followed by HRP-conjugated goat anti-mouse IgG. The membranewas developed using enhanced chemiluminescence.

[0014]FIG. 4. ATP synthase is present in rafts prepared from manydifferent cancer cell lines. Equal amounts of lipid raft proteinsprepared from various cancer cell lines (BeWo, Colo205, HT-29, JEG-3,KG-1, LS 180, MCF-7, LNCaP, NCI-H292, PANC-1, RT-4, and THP-1) wereseparated by SDS-PAGE and then electrotransferred onto a PVDF membrane.Western blotting was performed as described in FIG. 3.

[0015]FIG. 5. Flow cytometric analysis of ATP synthase cell surfaceexpression. LNCaP cells were resuspended in 100 μL PBS/5% fetal calfserum with 1 μg anti-ATP synthase (middle) or anti-Trop-1 (EpCAM)(right). Cells were also stained with a negative control antibody(left). Cells were washed and bound antibody was detected withPE-conjugated goat anti-mouse IgG. Cells were then analyzed by flowcytometer.

[0016]FIG. 6. Flow cytometric analysis of ATP synthase expression in theAML cell line THP-1. THP-1 cells were resuspended in 100 μL PBS/5% fetalcalf serum with 1 μg anti-ATP synthase α subunit (middle) or β subunit(right). Cells were also stained with a negative control antibody(left). Cells were washed and bound antibody was detected withPE-conjugated goat anti-mouse IgG. Cells were then analyzed by flowcytometer.

[0017]FIG. 7. ATP synthase is localized to the cell surface of LNCaPprostate carcinoma as visualized by immunofluorescence. LNCaP cells,grown on glass coverslips, were stained with antibodies specific for ATPsynthase (top) or Trop-1 (EpCAM) (bottom). Bound antibody was detectedwith Alexa 488-conjugated goat anti-mouse IgG. Coverslips were mountedonto slides and examined with a Nikon Optiphot 2 microscope andphotographed. No staining is observed on cells that have been stainedwith the secondary antibody alone (data not shown).

[0018]FIG. 8. Anti-ATP synthase inhibits LNCaP cell proliferation. LNCaPcells (20,000 cells/well) were plated into a 96 well tissue cultureplate. After cells were allowed to grow undisturbed for two days,antibodies (5 μg/ml anti-ATP synthase, anti-Trop-1 (EpCAM) (323/A3), oranti-MHC class II (Mu1D10)) were added and incubated with the cells for24 hours. AlamarBlue reagent was added to assess cell proliferation.Fluorescence was detected at λex=530 nm, λem=590 nm. Data are expressedas the mean+/−SEM of 4 replicates.

[0019]FIG. 9. Anti-ATP synthase inhibits LNCaP colony formation in softagar. LNCaP cells were plated in soft agar and treated with anti-ATPsynthase or anti-Trop-1 (EpCAM) (5 μg/ml) for up to 20 days. Colonieswere counted under an inverted phase-contrast microscope and a group of5 or more cells were counted as a colony.

[0020]FIG. 10. Anti-ATP synthase induces apoptosis in THP-1 cells. THP-1cells were treated with anti-ATP synthase or anti-Trop-1 (EpCAM) (5μg/mL) for 24 hours. Cells were then harvested at the indicated timesafter the induction of apoptosis and were stained with FITC-conjugatedannexin V and propidium iodide. Flow cytometry was used to assesspercentage of apoptosis (annexin V⁺ and propidium iodide^(−/+) cells).

[0021]FIG. 11. Comparison of lipid rafts from various cancer cell lines.Equal amounts of protein from lipid raft preparations were separated bySDS-PAGE and then visualized by silver staining.

[0022]FIG. 12. Table 1. shows five identified proteins from LNCaP lipidraft 2-D samples by peptide mass profiling.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Definitions:

[0024] As used herein, the term “antibody” or “immunoglobulin” refers toa protein consisting of one or more polypeptides substantially encodedby immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta, epsilon andmu constant region genes, as well as the myriad immunoglobulin variableregion genes. Full-length immunoglobulin “light chains” (about 25 Kd or214 amino acids) are encoded by a variable region gene at theNH2-terminus (about 110 amino acids) and a kappa or lambda constantregion gene at the COOH-terminus. Full-length immunoglobulin “heavychains” (about 50 Kd or 446 amino acids), are similarly encoded by avariable region gene (about 116 amino acids) and one of the otheraforementioned constant region genes, e.g., gamma (encoding about 330amino acids).

[0025] One form of immunoglobulin constitutes the basic structural unitof an antibody. This form is a tetramer and consists of two identicalpairs of immunoglobulin chains, each pair having one light and one heavychain. In each pair, the light and heavy chain variable regions aretogether responsible for binding to an antigen, and the constant regionsare responsible for the antibody effector functions. In addition toantibodies, immunoglobulins may exist in a variety of other formsincluding, for example, Fv, Fab, and (Fab′)₂, as well as bifunctionalhybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105(1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad.Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), which are incorporated herein by reference). (See, generally,Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), andHunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporatedherein by reference).

[0026] By “a pharmaceutically effective” amount of a drug orpharmacologically active agent or pharmaceutical formulation is meant anontoxic but sufficient amount of the drug, agent or formulation toprovide the desired effect.

[0027] A “subject,” “individual” or “patient” is used interchangeablyherein, which refers to a vertebrate, preferably a mammal, morepreferably a human.

[0028] The term “genetically altered antibodies” means antibodieswherein the amino acid sequence has been varied from that of a nativeantibody. Because of the relevance of recombinant DNA techniques to thisinvention, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

[0029] The term “humanized antibody” or “humanized immunoglobulin”refers to an immunoglobulin comprising a human framework, at least oneand preferably all complimentarily determining regions (CDRs) from anon-human antibody, and in which any constant region present issubstantially identical to a human immunoglobulin constant region, i.e.,at least about 85-90%, preferably at least 95% identical. Hence, allparts of a humanized immunoglobulin, except possibly the CDRs, aresubstantially identical to corresponding parts of one or more nativehuman immunoglobulin sequences. See, e.g. Queen et al., U.S. Pat. Nos.5,5301,101; 5,585,089; 5,693,762; and 6,180,370 (each of which isincorporated by reference in its entirety).

[0030] The term “chimeric antibody” refers to an antibody in which theconstant region comes from an antibody of one species (typically human)and the variable region comes from an antibody of another species(typically rodent).

[0031] The term “inhibit growth of cancer (tumor) cells” refers to anyaction that may decrease the growth of a cancer cell. The inhibition mayreduce the growth rate or the size of cancer cells, or inhibit orprevent proliferation, growth, or migration of cancer cells. Theinhibition may inhibit the colony formation of cancer cells due to theanchorage-independent growth. Preferably, such an inhibition at thecellular level may reduce the size, deter the growth, reduce theaggressiveness, or prevent or inhibit metastasis of a tumor (cancer) ina patient.

[0032] The term “colony formation” refers to the number of tumor(cancer) cell colonies formed due to the anchorage-independent tumor(cancer) cell growth. A variety of methods can be used to measure the“colony formation”, such as by counting the number of the formedcolonies formed (see Examples).

[0033] The present invention provides for a method of treating orpreventing prostate cancer.

[0034] In a preferred embodiment of the present invention, an inhibitorof ATP synthase activity is administered to a subject in need of such atreatment in a pharmaceutically effective amount.

[0035] The present invention also provides for a method of inhibitinggrowth of a cancer cell comprising contacting an inhibitor of ATPsynthase with said cancer cell. Preferably, said inhibiting reduces thecolony formation of a cancer cell by more than 50%, 60%, 70%, 80% or90%, or as high as 95%. More preferably, said cancer cell is a prostatecancer cell.

[0036] Preferably, the inhibitor is a protein.

[0037] More preferably, the protein directly interacts with ATPsynthase.

[0038] More preferably, the protein is an anti-ATP synthase antibody,including but not limited to an antibody recognizing the α-subunit ofATP synthase, or an antibody recognizing the β-subunit of ATP synthase.

[0039] Preferably, the anti-ATP synthase antibody can inhibit cancercell proliferation by more than 10%, more preferably by more than 20%.More preferably, said cancer cell is a prostate cancer cell.

[0040] Preferably, the anti-ATP synthase antibody can inhibit cancercell colony formation by more than 50%, 60%, 70%, 80% more preferably bymore than 90%, or by as high as about 95%. Preferably, said cancer cellis a prostate cancer cell.

[0041] The present invention provides a method of inducing apoptosis ofleukemia cells comprising contacting an antibody with said leukemiacells, wherein said antibody binds to or neutralizes ATP synthase.Preferably, said antibody induces apoptosis of the leukemia cells bymore than 20%. Preferably, said leukemia cells are acute myelogenousleukemia cells. More preferably, said leukemia cells are cells derivedfrom THP-1 cell line.

[0042] The present invention provides a method of treating eukemia in asubject in need of such a treatment comprising administering into saidsubject an antibody against ATP synthase in a pharmaceutically effectiveamount.

[0043] The anti-ATP synthase antibodies of the present invention may bein a polyclonal or monoclonal form and may bind to any epitope orsubunit of ATP synthase. Anti-ATP synthase antibodies of all species oforigins are included. Non-limiting exemplary anti-ATP synthaseantibodies include antibodies derived from human, chicken, goats, androdents (e.g., rats, mice, hamsters and rabbits), including transgenicrodents genetically engineered to produce fully human antibodies (see,e.g., Lonberg et al., WO93/12227 (1993) and Kucherlapati, et al.,WO91/10741 (1991)), which are herein incorporated by reference in theirentirety).

[0044] The polyclonal antibodies can be produced by immunization of hostanimals by ATP synthase. The polyclonal antibodies are secreted into thebloodstream and can be recovered using known techniques. Purified formsof these antibodies can, of course, be readily prepared by standardpurification techniques, preferably including affinity chromatographywith Protein A, anti-immunoglobulin, or the antigen itself. In any case,in order to monitor the success of immunization, the antibody levelswith respect to the antigen in serum will be monitored using standardtechniques such as ELISA, RIA and the like.

[0045] The monoclonal antibodies can be produced by conventionalhybridoma methodology known in the art. In particular, after theimmunization with ATP synthase, the host animal may be sacrificed andthe lymphocytes of said animal are isolated. The lymphocytes can produceor be capable of producing antibodies that specifically bind to theprotein used for immunization. Lymphocytes then are fused with myelomacells using suitable fusing agents to form hybridomas cells that producethe desired monoclonal antibody.

[0046] The antibodies may also be produced by using the method of lipidraft proteomics or lipid raft immunization, which is disclosed in U.S.Ser. No. 60/331,965, hereby incorporated by reference in its entirety.In particular, the method for identifying anti-tumor agents comprisesimmunizing an animal with lipid rafts from the interested cancer cells,such as prostate cancer cell, creating hybridomas from the immunizedanimal; screening the hybridomas, and purifying and identifying thehybridoma antibodies (see more details in U.S. Ser. No. 60/331,965).

[0047] The present invention also includes genetically alteredantibodies that are functionally equivalent to the anti-ATP synthaseantibodies. Modified antibodies providing improved stability and/ortherapeutic efficacy are preferred. Examples of modified antibodiesinclude those with conservative substitutions of amino acid residues,and one or more deletions or additions of amino acids which do notsignificantly deleteriously alter the antigen binding utility.Substitutions can range from changing or modifying one or more aminoacid residues to complete redesign of a region as long as thetherapeutic utility is maintained. Antibodies of this invention can bemodified post-translationally (e.g., acetylation, and phosphorylation)or can be modified synthetically (e.g., the attachment of a labelinggroup). The genetic modification can be achieved by the standardmolecular cloning techniques known in the art.

[0048] The genetically altered antibodies also include chimericantibodies that derived from the anti-ATP synthase antibodies.Preferably, the chimeric antibodies comprise a variable region derivedfrom a mouse or rat and a constant region derived from a human so thatthe chimeric antibody has a longer half-life and is less immunogenicwhen administered to a human subject. The method of making chimericantibodies is known in the art.

[0049] Preferably, the genetically altered anti-ATP synthase antibodiesused in the present invention include humanized version of theantibodies described herein. More preferably, said humanized antibodycomprising CDRs of a mouse donor immunoglobulin and heavy chain andlight chain frameworks of a human acceptor immunoglobulin. The method ofmaking humanized antibody is disclosed in U.S. Pat. Nos.: 5,530,101;5,585,089; 5,693,761; 5,693,762; 6,180,370, each of which isincorporated by reference in its entirety.

[0050] The fragments of the antibodies disclosed herein, which retainthe binding specificity to ATP Synthase, are also included in thepresent invention. Examples include, but are not limited to, the heavychains, the light chains, and the variable regions as well as thetruncated chains (truncated at the carboxyl end), which is particularlyuseful for immunoscintigraphic procedures. Examples of truncated chainsinclude, but are not limited to Fab fragment (consisting of the VL, VH,CL and CH1 domains); the Fd fragment (consisting of the VH and CH1domains); the Fv (consisting of VL and VH domains of a single arm of anantibody); dab fragment (consisting of a VH domain); isolated CDRregions; F(ab′)₂ fragment, a bivalent fragment (comprising two Fabfragments linked by a disulphide bridge at the hinge region). Thetruncated chains can be produced by conventional biochemistrytechniques, such as enzyme cleavage, or recombinant DNA techniques, eachof which is known in the art.

[0051] The variable region of the antibodies and its humanized versionof the present invention may be fused to functional regions from othergenes (e.g., enzymes, U.S. Pat. No. 5,004,692, which is incorporated byreference in its entirety) to produce fusion proteins (e.g.,immunotoxins) or conjugates having novel properties. When usedtherapeutically, the antibodies disclosed herein may be used inunmodified form or may be modified with an effector moiety that deliversa toxic effect, such as a drug, cytotoxin (preferably, a proteincytotoxin or a Fe domain of the monoclonal antibodies), radionuclide,etc (see, e.g., U.S. Pat. No. 6,086,900, which is incorporated byreference in its entirety).

[0052] Conjugates that are immunotoxins including conventionalantibodies have been widely described in the art. The toxins may becoupled to the antibodies by conventional coupling techniques orimmunotoxins containing protein toxin portions can be produced as fusionproteins. The conjugates of the present invention can be used in acorresponding way to obtain such immunotoxins. Illustrative of suchimmunotoxins are those described by Byers, B. S. et al. Seminars CellBiol 2:59-70 (1991) and by Fanger, M. W. et al. Immunol Today 12:51-54(1991). (See, generally, “Chimeric Toxins,” Olsnes and Phil, Pharmac.Ther., 25, 355-381 (1982), and “Monoclonal Antibodies for CancerDetection and Therapy,” eds. Baldwin and Byers, pp. 159-179, 224-266,Academic Press (1985),

[0053] In addition, the inhibitor can also be a compound that inhibitsthe enzyme activity of ATP synthase, including but not limited, to anantagonist for ATP synthase. More examples include compounds that mayinteract with ATP synthase signaling pathway and down-regulates theactivity of ATP synthase. The understanding of the mechanism of ATPsynthase in tumor activity provides the useful information for thesearch of this kind of compounds. Several existing theories explain thepossible role of ATP synthase on tumor cell surface. First of all,cell-surface-generated ATP may be transported into the cell to provide asource of energy in the tumor microenvironment where Po₂ levels are verylow. Alternatively, cell surface-generated ATP may act through the P2Yreceptor to activate Ca²⁺-dependent signaling cascades which increaseDNA synthesis. The third theory is that ATP synthase may pump outprotons which in turn acidifies the tumor microenvironment and as aconsequence, pH-dependent enzymes produced by tumor cells are able tocut through the extracellular matrix and metathesize. Any of thesetheories provides a starting point to search for the ideal compounds ormolecules as ATP synthase inhibitor. The suitable compounds can besought by using the conventional techniques known to a skilled artisanin the field of organic chemistry and biochemistry.

[0054] In addition, the inhibitor can inhibit the protein expression ofATP synthase. The inhibitor is a nucleic acid including but not limitedto an antis-sense nucleic acid of the nucleic acid sequence encodingpart or full or having substantial sequence similarity of ATP synthase.The DNA sequence of ATP synthase is known in the art. Subsequently,anti-sense nucleic acid probe of ATP synthase DNA, and the optimalcondition of the anti-sense blocking can be developed by using therelated techniques known to a skilled artisan in the field of molecularbiology.

[0055] The present invention also provides for a pharmaceuticalcomposition comprising an inhibitor of ATP synthase activity. Thepharmaceutical composition can further comprise a pharmaceuticallyacceptable carrier.

[0056] The effective treatment of prostate cancer by ATP synthaseinhibitors can include various stages, such as androgen-dependentprostate cancer and androgen-independent prostate cancer.

[0057] For the purpose of treatment of disease, the appropriate dosageof the above inhibitors will depend on the severity and course ofdisease, the patient's clinical history and response, the toxicity ofthe inhibitors, and the discretion of the attending physician. Theinhibitors are suitably administered to the patient at one time or overa series of treatments. The initial candidate dosage may be administeredto a patient. The proper dosage and treatment regime can be establishedby monitoring the progress of therapy using conventional techniquesknown to the people skilled of the art.

[0058] Dosage levels of the order of from about 10⁻⁷ M to about 10⁻¹ M,preferably in the range 10⁻⁵ to 10⁻¹M, are useful in the treatment. Thepharmaceutically effective amount ranges between about 0.01 to about1000 mg, preferably between about 0.05 to about 100 mg, and mostpreferably between about 0.5 to about 50 mg for single doses. The amountof active ingredients that may be combined with the carrier materials toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. It will be understood,however, that the specific dose level for any particular patient willdepend upon a variety of factors, including the activity of the specificinhibitor employed, the age, body weight, general health, sex, diet,time of administration, route of administration, and rate of excretion,drug combination and the severity of the particular disease undergoingtherapy, and can be determined by those skilled in the art.

[0059] There are various methods of administering the inhibitors. Thereare various methods of administering the inhibitors. The inhibitor maybe administered to a patient intravenously as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, inhalation routes, or other delivery meansknown to the people skilled in the art.

[0060] Preferably, pharmaceutical compositions of the present inventionare useful for parenteral administration, i.e., subcutaneously,intramuscularly and particularly, intravenously. The compositions forparenteral administration commonly comprise a solution of the inhibitorof ATP synthase, preferably the anti-ATP synthase antibody, or acocktail thereof dissolved in an acceptable carrier, preferably anaqueous carrier. A variety of aqueous carriers can be used, e.g., water,buffered water, 0.4% saline, 0.3% glycine and the like. These solutionsare sterile and generally free of particulate matter. The compositionsmay contain pharmaceutically acceptable auxiliary substances as requiredto approximate physiological conditions such as pH adjusting andbuffering agents, toxicity adjusting agents and the like, for examplesodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate, histidine and arginine. The concentration of theinhibitors (preferably antibodies) in these formulations can varywidely, i.e., from less than about 0.01%, usually at least about 0.1% toas much as 5% by weight and are selected primarily based on fluidvolumes, and solubilities in accordance with the particular mode ofadministration selected.

[0061] The present invention also provides for a method of detectingprostate cancer, comprising detecting the presence of ATP synthase inthe prostate cells of a subject in need of such detection. The presentinvention also provides a method of detecting breast cancer, coloncancer, or leukemia (preferably, acute myelogenous leukemia) comprisingdetecting presence of ATP synthase in the breast cells, colon cells, andT-cells respectively. Since our data show that ATP synthase isdifferentially expressed on the surface of the prostate cancer cells-,and leukemia cells but not in normal cells, an antibody against ATPsynthase can be used as a bio-marker for detecting these types ofcancers. The antibody includes, but is not limited to, the antibodyagainst α-subunit of ATP synthase.

[0062] The present invention also provides for a diagnostic kitcomprising anti-ATP synthase antibodies. Such a diagnostic kit furthercomprises a packaged combination of reagents in predetermined amountswith instructions for performing the diagnostic assay. Where theantibody is labeled with an enzyme, the kit will include substrates andco-factors required by the enzyme. In addition, other additives may beincluded such as stablizers, buffers and the like. The relative amountsof the various reagents may be varied widely to provide forconcentrations in solution of the reagents that substantially optimizethe sensitivity of the assay. Particularly, the reagents may be providedas dry powders, usually lyophilized, including excipients that, ondissolution, will provide a reagent solution having the appropriateconcentration.

[0063] The inhibitors of the present invention may also be employed forthe inhibition of cancer cell growth, or for the treatment of othertypes of cancer or neoplasm or malignant tumors found in mammals,including carcinomas and sarcomas. Examples of cancers are cancer of thebrain, breast, cervix, bladder, colon, head & neck, kidney, lung,non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach,uterus, and medulloblastoma. Preferably, the inhibitors may be employedto detect or treat disorders including, but not limited to, Hodgkin'sDisease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breastcancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primarythrombocytosis, primary macroglobulinemia, small-cell lung tumors,primary brain tumors, stomach cancer, colon cancer, malignant pancreaticinsulanoma, malignant carcinoid, urinary bladder cancer, premalignantskin lesions, testicular cancer, lymphomas, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, cervical cancer, endometrial cancer, adrenal corticalcancer, and leukemia.

[0064] Though the inhibitors of the present invention are primarilyconcerned with the treatment of human subjects, they may also beemployed for the treatment of other mammalian subjects such as dogs andcats for veterinary purposes.

[0065] Additionally, the inhibitors can be utilized alone insubstantially pure form, or together with chemotherapeutic agents, asare known to those of skill in the art (see, e.g., Cancer: Principlesand Practice of Oncology, 5^(th) ed., Devita et al., Lippincott-RavelPublishers, 1997). Other therapies that may be used in conjunction withtreatment with the antibodies include administration of anti-sensenucleic acid molecules or biologicals, such as additional therapeuticantibodies, as well as radiation and/or surgery (see, e.g., WO0034337).Thus, the treatment of the present invention is formulated in a mannerallowing it to be administered serially or in combination with anotheragent for the treatment of cancer.

[0066] The following examples are offered by way of illustration and notby way of limitation. The disclosure of all citations in thespecification is expressly incorporated herein by reference.

EXAMPLES Example 1

[0067] This example describes the identification of anti-tumor targetsfor the treatment of prostate cancer by using lipid raft proteomics.

[0068] In the present invention, molecules critical to the treatment ofprostate cancer were sought by initially detecting differentialexpression of proteins in normal cells and the prostate tumor celllines. Lipid rafts of each cell line were isolated and studiedsubsequently.

[0069] Materials and Methods

[0070] a. Lipid Raft Preparation

[0071] Lipid rafts were prepared as described in Green et al, J. CellBiol. 146, 673-682 (1999). Briefly, cells (8.0×10⁶ cells/sample) werelysed in 0.1% vol/vol Brij-58, 20 mM Tris HCl, pH 8.2, 140 mM NaCl, 2 mMEDTA, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 1 mMphenylmethylsulfonyl fluoride for 10 minutes on ice. Cells werehomogenized using 10 strokes of a Dounce homogenizer, then lysed 20minutes more on ice. The resulting lysate was adjusted to 40% wt/wtsucrose and applied onto a 60% wt/wt sucrose cushion. A sucrosestep-gradient consisting of 25% wt/wt sucrose and 5% wt/wt sucrose werelayered on top of the lysate. Gradients were centrifuged 18 hours at170,000×g at 4° C. in a SW55 rotor. Fractions (0.2 ml) were taken fromthe top of the gradient. Lipid rafts float to the interface of the 25%and 5% sucrose layers (Fractions 7 and 8). The amount of protein in eachfraction was determined using the BCA Protein Assay Kit. Protein wasconcentrated by centrifugation at 2000×g in Vivaspin 6 PES membranecolumns (molecular weight cut off=10,000 kDa).

[0072] b. Electrophoresis and Western Blotting

[0073] Lipid raft proteins were separated by SDS-PAGE on a 4-20%gradient gel and then electrotransferred onto a polyvinylidenedifluoride membrane (PVDF). The membrane was blocked for 1.5 hours atroom temperature in PBS with 5% milk. The membrane was then incubatedwith 0.4 μg/ml mouse anti-ATP synthase (Molecular Probes, catalog#A-11144) in PBS with 1% BSA and 0.5% Tween-20 for 2 hours at roomtemperature. After extensive washing, the membrane was incubated withHRP-conjugated goat antibodies specific for mouse IgG for 1 hour at roomtemperature in PBS with 1% BSA and 0.5% Tween-20. After extensivewashing, blot was developed using enhanced chemiluminescence followed byfluorography.

[0074] c. N-terminal Sequencing

[0075] Proteins to be sequenced were separated by SDS-PAGE on a 4-20%gradient gel and then electrotransferred onto a PVDF membrane. Themembrane was stained for 2 minutes using colloidal Coomassie and thendestained in water. The resulting bands were excised and subjected toN-terminal Edman sequencing as described by Miller, Methods: A Companionto Methods in Enzymology 6, 315 (1994). Results were confirmed usingmatrix assisted laser desorption ionization-time of flight (MALDI-TOF)peptide-mass profiling.

[0076] d. MALDI-TOF Peptide-mass Profiling

[0077] Proteins to be analyzed were separated by SDS-PAGE on a 4-20%gradient gel. Alternatively, 2-dimensional electrophoresis was used tofurther separate lipid raft proteins using an IPGphor IsoelectricFocusing System according to the manufacturers protocol (AmershamPharmacia Biotech, Piscataway, N.J.). Proteins were visualized bystaining the resulting gel with 0.05% Coomassie Blue R250, 50% methanol,10% acetic acid in water followed by destaining in 15% methanol, 10%acetic acid in water.

[0078] Protein band or spot of interest was excised with a razor bladeand equilibrated in 100 mM Tris HCl, pH 8.5 at room temperature for 45minutes. The solution was replaced with 150 μL of 2 mM DTT in 100 mMTris-HCl, pH 8.5. The samples were incubated with agitation for 30minutes at 60° C. The solution was replaced with 150 82 L of 20 mMiodoacetic acid in 100 mM Tris-HCl, pH 8.5. The samples were incubatedin the dark at 37° C. for 30 minutes. The solution was replaced with 150μL of equal parts 100 mM Tris-HCl, pH 8.5 and acetonitrile. The tubeswere shaken vigorously at 37° C. for 45 minutes. This step was repeateduntil the gel bands were clear. The solution was removed and the gelslices were dried in a SpeedVac on low vacuum strength for 15 minutes.The gel bands were re-swelled with 0.25-0.5 μg of a concentratedendo-protease Lysine-C or trypsin solution, then covered with 50-80 μLof 100 mM Tris-HCl, pH 8.5, 10% acetonitrile and incubated withagitation for 18 hours at 37° C. After digestion the samples were storedat 4-8° C.

[0079] The digest solution was removed from the micro-centrifuge tube,acidified with 10% trifluoroacetic acid (TFA) in water v/v to 1% TFAv/v, desalted and concentrated using a C18 Zip-Tip. The micro-columneluate was combined 1:1 with 10 mg/ml alpla-Cyano-4-hydroxycinamic acidin 60% acetonitrile and spotted on a MALDI-TOF sample plate. A closeexternal calibrant with the approximate concentration of the sample wasspotted adjacent to the sample position on the MALDI plate. Thecalibrant was prepared by diluting a pre-made calibration mix consistingof angiotensin II fragment 1-7 and adrenocorticotropic hormone fragment18-39 with Zip-Tip eluant. The diluted calibration mixture was combined1:1 with matrix solution. The sample and calibrant spots were driedsimultaneously at room temperature.

[0080] MALDI mass fingerprints were acquired on a Perceptive BiosystemsVoyager DE Pro MALDI-TOF in reflector mode with delayed extraction andpositive polarity. Approximately 100-300 shots from a 20 KV laser wereaccumulated. During spectrum acquisition a resolution calculator wasemployed to ensure accurate calibration. After the spectra were acquiredand calibrated, the monoisotopic masses were automatically selectedusing the de-isotoping function on Voyager Software. The calibratedmonoisotopic peak lists were exported into ProteinProspector MS-Fitversion 3.2.1 and searched against the largest non-redundant databaseavailable from the National Center for Biotechnology Information(NCBInr).

[0081] e. Flow Cytometry Staining

[0082] Flow cytometry was used to screen hybridoma supernatants for thepresence of cell surface binding antibodies. The cells (2×10⁵) wereresuspended in 100 μL ice cold PBS with 10 μL tissue culture supernatanton ice for 1 hour. After extensive washing, cells were incubated withphycoerythrin-conjugated goat antibodies specific for mouse IgG for 30minutes on ice. Cells were washed again and cell surface bound antibodywas detected using a Becton Dickenson FACScan. Additionally, hybridomasupernatants were similarly screened on many cancer cell lines or wholeblood to test for specificity.

[0083] f. Immunofluorescence

[0084] LNCaP cells were grown on glass coverslips undisturbed for twodays. Cells were fixed with 3% paraformaldehyde in PBS for 15 minutes.After being washed with PBS, cells were incubated in 50 mM NH₄Cl in PBSfor 10 minutes. After washing with PBS, cells were subsequentlyincubated with 5% goat serum in PBS for 30 minutes followed by stainingwith 5 μg/ml anti-ATP synthase or anti-Trop-1 (EpCAM) in 2.5% goat serumin PBS for 1 hour at room temperature. Cells were stained with anti-MHCclass II (Kostelny et al Int. J. Cancer 93, 556 (2001)) as a negativecontrol. After washing with PBS, bound antibody was detected byincubating cells with Alexa 488-conjugated goat antibodies specific formouse IgG in 2.5% goat serum in PBS for 30 minutes at room temperature.After extensive washing, glass coverslips were mounted in a solutioncontaining Mowiol 4-88, glycerol, and 150 mM Tris HCl, pH 8.5. Slideswere placed at 4° C. overnight before viewing. Cells were analyzed byfluorescence microscopy on a Nikon Optiphot 2 microscope.

[0085] g. LNCaP Proliferation

[0086] LNCaP cells were plated at 20,000 cells/well into a 96 welltissue culture plate. After cells were allowed to grow undisturbed fortwo days, antibodies (5 μg/ml anti-ATP synthase, anti-Trop-1 (EpCAM), oranti-MHC class II) were added and incubated with the cells for 24khours. Cell proliferation was measured using the AlamarBlue vital dyeindicator assay. AlamarBlue reagent was added to each well and theplates were incubated for 3 to 4 hours at 37° C. to allow forfluorescence development. Fluorescence was detected at λex=530 nm,λem=590 nm. Data are expressed as the mean+/−SEM of 4 replicates.

[0087] h. Soft Agar Colony Formation Assay

[0088] For anchorage-independent cell growth, a soft agar colonyformation assay was performed in a six-well plate. Each well contained 2mL of 1% agar in complete medium as the bottom layer. The top layercontained 2 mL 0.5% agar in complete medium, 1000-10000 LNCaP cells, and5 μg/mL mAb (anti-ATP synthase, or anti-Trop-1). One mL complete mediumwas added and the cultures were maintained at 37° C. in a humidified 5%CO₂ atmosphere for up to 20 days. One mL complete medium was added oncea week. Media was removed and the colonies were stained with 0.005%crystal violet in PBS for 2 hours. The number of colonies was determinedby counting them under an inverted phase-contrast microscope at 100×,and a group of 10 or more cells were counted as a colony.

[0089] Results and Discussion

[0090] a. Differential Expression of ATP Synthase in Prostate TumorCells

[0091] Lipid rafts were extracted from normal prostate cells and threewidely used prostate cancer cells—LNCaP, DU145, and PC-3. The proteinexpression of each sample was compared using one-dimensionalelectrophoresis together with silver staining. Several protein bands inthe one-dimensional electrophoresis gel appeared only in the prostatecancer cell lines, indicating that they are candidate proteins relatedto the prostate cancer. One of the candidate proteins with molecularweight of approximately 50 kD (denoted by an arrow with a “*” in FIG. 1)was selected for N-terminal sequencing. The sequencing result indicatedthat it was the β-subunit of ATP synthase.

[0092] The differential protein expression was also confirmed bytwo-dimensional electrophoresis and then visualized by silver staining.Protein spots that are present in the LNCaP sample, but not the normalprostate sample are denoted with arrows (16 spots identified), as shownin FIG. 2. Many of these lipid raft proteins were excised and subjectedto MALDI-TOF peptide mass profiling analysis. The identities of 5 ofthem are shown in Table 1. The locations of these proteins on the 2-Dgel are labeled with numbers as shown in FIG. 2. We found 2 subunits ofATP synthase (α and β subunits), 2 voltage-dependent anion channel/porinproteins, adenine nucleotide translocator, and prohibitin. All of these5 identified proteins are mitochondria proteins but somehow areassociated with lipid rafts of LNCaP cells. It is not known how many ofthese lipid raft-associated proteins are exposed to the outer surface ofthe cell membrane and therefore are accessible to antibodies. As severalanti-ATP synthase antibodies are commercially available, we used one ofthem to confirm that some ATP synthase molecules are located in lipidrafts and they are accessible to antibodies.

[0093] Western blot analysis of lipid rafts using anti-α-subunit ATPsynthase antibody further confirmed the correlation between the ATPsynthase and prostate cancer. As shown in FIG. 3, positive stainingappeared in all three prostate cancer cell lines, but not the normalcells, indicating that ATP synthase was present in lipid rafts ofprostate cancer cells but not normal cells. In addition, similar Westernblot analysis of lipid rafts from cancer cells of different originsindicates that some cancer cell lines, such as the AML cell lines KG-1and THP-1, the breast cancer cell line MCF-7, the colon cancer cell lineLS180, and the bladder cancer cell line RT4 also express ATP synthase inlipid rafts (FIG. 4). ATP synthase was also showed to be expressed onthe surface of the androgen-independent cell line DU 145 by a similaranalysis.

[0094] b. Cell Surface Localization of ATP Synthase

[0095] To investigate the surface localization of the ATP synthase,prostate tumor cell line LNCaP was analyzed by FACS staining andimmunofluorescence microscopy. As shown in FIG. 5, FACS staining byanti-ATP synthase antibody in LNCaP tumor cells revealed about 25.9% ofcells had ATP synthase cell surface expression, Staining with anantibody against Trop-1 (EpCAM), a cell surface protein expressed oncancer cells, showed surface localization in 92.8% of the cells. Similaranalysis of the AML cell line THP-1 by flow cytometry also showed thatthese cells have ATP synthase on their cell surface (FIG. 6).

[0096] The surface localization of ATP synthase was further evidenced bythe immunofluorescence microscopy. As shown in FIG. 7, immunostaining ofLNCaP cells with anti-ATP synthase gave rise to positive cell surfacestaining. The colocalization of Trop-1 (EpCAM) and ATP synthaseindicated that ATP synthase was indeed expressed on the surface ofprostate tumor cells.

[0097] Moreover, FACS staining of LNCaP tumor cells growing in Matrigel®demonstrated a much higher percentage of cells exhibiting a positivesurface staining of ATP synthase, confirming that ATP synthase cellsurface expression can be modulated by cellular environment, and thismay play important roles in the real biological environment (data notshown).

[0098] c. Inhibition of Prostate Cancer Cell Proliferation in thePresence of Anti-ATP Synthase

[0099] As shown in FIG. 8, LNCaP tumor cell proliferation can be reducedby antibodies specific for ATP synthase. This reduction in cellularproliferation ranges from 12.5% to 27.8%. This substantial inhibition ofcell proliferation suggests a potential of anti-ATP synthase antibody asan anti-tumor agent for treating prostate cancer.

[0100] The above experiments demonstrate that ATP synthase is closelyrelated to the prostate cancer cells. It is localized on the surface ofprostate tumor cells, and may play a key role in the biologicalactivities of prostate cancer cells. Blocking the activity of ATPsynthase by antibodies inhibits the prostate tumor cell growth,suggesting the possibility of clinical application of ATP synthaseinhibitors as anti-tumor agents for treating prostate cancer.

[0101] d. Inhibition of Prostate Cancer Cell Colony Formation in SoftAgar in the Presence of Anti-ATP Synthase

[0102] Transformed cancer cells are resistant to anchorage-independentgrowth inhibition and are able to growth in soft agar without attachingto cell matrix. Formation of colonies (three-dimensional growth undertissue culture growth conditions) of cancer cells in soft agar is oftencorrelated to the aggressiveness of the tumor in vivo. To assess whetheranti-ATP synthase has any anti-cancer activity, we used it inhibit LNCaPcolony formation in vitro. As shown in FIG. 9, LNCaP colony formationcan be reduced by an antibody specific for ATP synthase (anti-α subunit)at 5 μg/ml. This reduction in colony formation by anti-ATP synthasecould be as high as 95%, whereas the action of an anti-Trop-1 antibody(Ep-CAM) antibody was less impressive, at about 68%. This substantialinhibition of colony formation suggests a potential of anti-ATP synthaseantibody as an anti-tumor agent for treating prostate cancer.

[0103] e. AML Cell Death Induction in the Presence of Anti-ATP Synthase

[0104] The anti-cancer activity of anti-ATP synthase can also bedemonstrated in the AML cell line THP-1, which expresses ATP synthase onthe cell surface. Incubation of THP-1 cells with anti-ATP synthase at 5μg/ml for 24 hours led to substantial cell death as assayed by flowcytometry (FIG. 10). Compared to PBS or an irrelevant antibody control,the specific killing induced by anti-ATP synthase was 25%. This anti-AMLactivity suggests a potential of antiATP synthase antibody asanti-leukemia or lymphoma agent to treat hematological malignancies.

[0105] The above experiments demonstrate that ATP synthase expression isclosely related to certain cancer cells. It is localized on the surfaceof prostate and AML cancer cells, and may play a key role in thebiological activities of prostate and AML cancer cells. Blocking theactivity of ATP synthase by antibodies inhibits the prostate cancer cellgrowth and colony formation, and induces cell death in AML cancer cells,suggesting the possibility of clinical application of ATP synthaseinhibitors as anti-tumor agents for treating prostate cancer and AML.

Example 2

[0106] This example describes the treatment of prostate cancer byantibodies specific for ATP synthase in well-establishedandrogen-dependent and androgen-independent prostate cancer xenografts.

[0107] Six to ten week old male nude NCR nu/nu mice are inoculatedsubcutaneously in the mid-scapular region with 5×10⁶ androgen-dependentLNCaP cells. Cells that are injected are reconstituted with basementmembrane in the form of Matrigel as described (Sato et al Cancer Res. 5,1584-1589 (1997)). To maintain serum testosterone levels, male mice areimplanted with 12.5 mg sustained release testosterone pelletssubcutaneously prior to receiving the tumor cell inoculation. Antibodiesspecific for ATP synthase (Molecular Probes, cat #A-11144 and A-21299)are given intraperitoneally on day 2 and 4. Tumors are measured everythree to four days with vernier calipers. Tumor volumes are calculatedby the formula π/6×(larger diameter)×(smaller diameter)². For theandrogen-independent prostate cancer xenograft studies, DU 145 or PC-3cells are used.

Example 3

[0108] This example describes the human prostate cancer therapeuticregime by using the inhibitors of ATP synthase.

[0109] The patient's cancer biopsy sample is stained positively for theexpression of ATP synthase on the cancer cell surface byimmunohistochemistry for the patient to be eligible for treatment.Anti-ATP synthase antibodies are administered either intravenously orsubcutaneously in a dose range from 0.05 to about 25 mg/kg. Patientsreceive at least 4 weekly doses. Tumor size is monitored by CAT scan orMRI prior to therapy and post therapy. Reduction of the tumor size isthe primary indication of the drug's efficacy. Tumor shrinkage by 50% ormore is considered as a partial response. Complete disappearance of thetumor is considered as a complete response. For prostate cancerpatients, PSA level prior to therapy and post therapy is also monitoredas a secondary indication of treatment efficacy.

[0110] Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention.

[0111] All publications, patents, patent applications, and web sites areherein incorporated by reference in their entirety to the same extent asif each individual patent, patent application, or web site wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A method of treating or preventing prostate cancer in a subject inneed of such a treatment or prevention comprising administering intosaid subject an inhibitor of ATP synthase activity in a pharmaceuticallyeffective amount.
 2. The method according to claim 1, wherein saidinhibitor inhibits protein expression of ATP synthase.
 3. The methodaccording to claim 1, wherein said inhibitor is an anti-sense nucleicacid of a nucleic acid sequence encoding part or full ATP synthase. 4.The method according to claim 2, wherein said inhibitor down-regulatesbiological activities of ATP synthase.
 5. The method according to claim1, wherein said inhibitor is a protein that directly interacts with ATPsynthase.
 6. The method according to claim 5, wherein said inhibitor isan anti-ATP synthase antibody.
 7. The method according to claim 6,wherein said antibody is a monoclonal antibody.
 8. The method accordingto claim 6, wherein said antibody inhibits prostate cancer cellproliferation by at least 10%.
 9. The method according to claim 7,wherein said antibody inhibits prostate cancer cell proliferation byabout 20%.
 10. The method according to claim 6, wherein said antibodyinhibits prostate cancer cell colony formation by at least 50%.
 11. Themethod according to claim 10, wherein said antibody inhibits prostatecancer cell colony formation by about 95%.
 12. A pharmaceuticalcomposition comprising an inhibitor of ATP synthase.
 13. A method ofdetecting prostate cancer comprising detecting presence of ATP synthasein prostate cells of a subject in need of such detection.
 14. The methodaccording to claim 7, wherein said monoclonal antibody is a humanizedantibody or a fully human antibody.
 15. The method according to claim 7,wherein said monoclonal antibody is a chimeric antibody.
 16. The methodaccording to claim 7, wherein said antibody is an antibody tetramer,Fab, (Fab′)₂, or Fv.
 17. The method according to claim 1, wherein saidinhibitor is an antibody conjugate comprising an anti-ATP synthaseantibody.
 18. The method according to claim 17, wherein said anti-ATPsynthase antibody is conjugated to a cytotoxin agent.
 19. The methodaccording to claim 18, wherein said cytotoxin agent is a proteincytotoxin or a Fc domain of a monoclonal antibody.
 20. The methodaccording to claim 1, further comprising administering achemotherapeutic agent to the subject, wherein said treating isformulated in a manner allowing it to be administered serially or incombination with another agent for treatment of cancer.
 21. A method ofinhibiting growth of a cancer cell comprising contacting an inhibitor ofATP synthase with said cancer cell.
 22. The method according to claim21, wherein said cancer cell is a prostate cancer cell.
 23. The methodaccording to claim 22, wherein said inhibitor is an antibody that bindsto or neutralizes ATP synthase.
 24. The method according to claim 23,said antibody reduces colony formation of said prostate cancer cell byat least 50%.
 25. The method according to claim 24, said antibodyreduces colony formation of said prostate cancer cell by about 95%. 26.A method of inducing apoptosis of leukemia cells comprising contactingan antibody with said leukemia cells, wherein said antibody binds to orneutralizes ATP synthase.
 27. The method according to claim 26, whereinsaid leukemia cells are acute myelogenous leukemia cells.
 28. The methodaccording to claim 26, wherein said leukemia cells are cells derivedfrom THP-1 cell line.
 29. A method of treating leukemia in a subject inneed of such a treatment comprising administering into said subject anantibody against ATP synthase in a pharmaceutically effective amount.30. A method of detecting breast cancer comprising detecting presence ofATP synthase in breast cells of a subject in need of such detection. 31.A method of detecting colon cancer comprising detecting presence of ATPsynthase in colon cells of a subject in need of such detection.
 32. Amethod of detecting leukemia comprising detecting presence of ATPsynthase in T-cells of a subject in need of such detection.
 33. Themethod according to claim 32, wherein said leukemia is acute myelogenousleukemia.